Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation.

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.

Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.

DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20-30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling.

GPT-4 as a biomedical simulator.

BACKGROUND: Computational simulation of biological processes can be a valuable tool for accelerating biomedical research, but usually requires extensive domain knowledge and manual adaptation. Large language models (LLMs) such as GPT-4 have proven surprisingly successful for a wide range of tasks. This study provides proof-of-concept for the use of GPT-4 as a versatile simulator of biological systems. METHODS: We introduce SimulateGPT, a proof-of-concept for knowledge-driven simulation across levels of biological organization through structured prompting of GPT-4. We benchmarked our approach against direct GPT-4 inference in blinded qualitative evaluations by domain experts in four scenarios and in two quantitative scenarios with experimental ground truth. The qualitative scenarios included mouse experiments with known outcomes and treatment decision support in sepsis. The quantitative scenarios included prediction of gene essentiality in cancer cells and progression-free survival in cancer patients. RESULTS: In qualitative experiments, biomedical scientists rated SimulateGPT's predictions favorably over direct GPT-4 inference. In quantitative experiments, SimulateGPT substantially improved classification accuracy for predicting the essentiality of individual genes and increased correlation coefficients and precision in the regression task of predicting progression-free survival. CONCLUSION: This proof-of-concept study suggests that LLMs may enable a new class of biomedical simulators. Such text-based simulations appear well suited for modeling and understanding complex living systems that are difficult to describe with physics-based first-principles simulations, but for which extensive knowledge is available as written text. Finally, we propose several directions for further development of LLM-based biomedical simulators, including augmentation through web search retrieval, integrated mathematical modeling, and fine-tuning on experimental data.

EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells.

Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.

Epigenetic Modulation of Radiation-Induced Diacylglycerol Kinase Alpha Expression Prevents Pro-Fibrotic Fibroblast Response.

Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis.

Simple Synthesis of Functionalized Paramagnetic Beads for Nucleic Acid Purification and Manipulation.

The purification of nucleic acids is one of the most common procedures employed in modern molecular biology laboratories. Typically, commercial column-based protocols are utilized to isolate DNA or RNA from various sources. However, these methods not only require specialized equipment, but are also extremely expensive for high-throughput applications. Although an elegant answer to this issue can be provided by paramagnetic beads, bead-based open-source protocols have been limited in the past. Here, we provide an easy to follow step-by-step manual for the synthesis of paramagnetic beads, as well as their functionalization with either a silica- or a carboxyl-surface that can be used to replace the commercial columns with self-made magnetic beads. Together with a variety of detailed protocols for their use in high-throughput nucleic acids extractions, this bead synthesis method forms the recently published open platform Bio-On-Magnetic-Beads (BOMB), which is available on PLOS Biology ( Oberacker et al., 2019 ). Updated protocols can be found on the associated webpage (https://bomb.bio).

Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors.

Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation is not clear. To investigate this question, we use a CRISPR-dCas9 epigenetic editing tool, where an inactive form of Cas9 is fused to DNA methyltransferase effectors. Using this system, here we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary breast cells isolated from healthy human breast tissue. We find that promoter methylation is maintained in this system, even in the absence of the fusion construct, and this prevents cells from engaging senescence arrest. Our data show that the key driver of this phenotype is repression of CDKN2A transcript p16 where myoepithelial cells harbour cancer-like gene expression but do not exhibit anchorage-independent growth. This work demonstrates that hit-and-run epigenetic events can prevent senescence entry, which may facilitate tumour initiation.

Synthetic epigenetics-towards intelligent control of epigenetic states and cell identity.

Epigenetics is currently one of the hottest topics in basic and biomedical research. However, to date, most of the studies have been descriptive in nature, designed to investigate static distribution of various epigenetic modifications in cells. Even though tremendous amount of information has been collected, we are still far from the complete understanding of epigenetic processes, their dynamics or even their direct effects on local chromatin and we still do not comprehend whether these epigenetic states are the cause or the consequence of the transcriptional profile of the cell. In this review, we try to define the concept of synthetic epigenetics and outline the available genome targeting technologies, which are used for locus-specific editing of epigenetic signals. We report early success stories and the lessons we have learned from them, and provide a guide for their application. Finally, we discuss existing limitations of the available technologies and indicate possible areas for further development.